Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne infection caused by Anaplasma (Ehrlichia) phagocytophila, an obligate intracellular bacteria that infects neutrophils. The infection is widespread and severity ranges from fatal to subclinical; the seroprevalence reaches 15 percent in some areas. We characterized antigenic variation in the major immunodominant surface protein (Msp) of the bacterium that likely results from differential transcription of one of at least 18 different gene copies. Moreover, we also recently discovered that pathogenicity in a murine model is due to induction of immunity, chiefly interferon-gamma, and is abrogated by interleukin-10, despite the presence of large numbers of bacteria. Because of these observations, we hypothesize that induction of pathologic lesions and disease depend upon immunity that varies with changing bacterial factors, possibly Msp. To test this hypothesis and to more completely understand pathogenetic mechanisms of granulocytic ehrlichiae, we will use a convenient mouse model and validate results in the horse model that mostly closely simulates human disease. Thus, we propose to: 1. Characterize the diversity and induce changes in expression of A. phagocytophila major surface protein genes in vitro and in vivo. Partial Msp cDNA transcripts from infected humans and animals will be sequenced and compared to identify evidence of ongoing gene transcriptional changes, and laboratory strains will be induced to alter the expressed Msp by specific-Msp antibodies, prolonged in vitro cultivation, or in vivo infection. 2. Demonstrate differences in pathology and immune responses after challenge with virulent (low passage) and reduced virulence (high passage) A. phagocytophila. Mice and horses infected with strains of ehrlichiae that differ in virulence will be assessed for T cell and cytokine phenotype and results will be correlated with clinical signs and pathology. 3. Demonstrate that innate immune responses to whole ehrlichiae and component Msps comprise part of the early proinflammatory response. Innate responses to native components of virulent and attenuated virulence ehrlichiae will be assessed for their ability to initiate inflammatory and immune reactions via toll- like receptors (TLRs), IL-12;, IL-18, and NK cells in vitro and in vivo in knockout mice. Immunophenotype and cytokine profiles will be correlated with clinical signs or pathologic lesions in the infected animal models for both. 4. Characterize how the components of the adaptive immune response to whole ehrlichiae and component Msp influence the histopathology and clinical signs in GE. The role of specific immune cells (CD4, CD8, and NK) as well as specific effector mechanisms (nitric oxide, reactive oxygen intermediates, perforin, complement) that can lead to cell injury, histopathology, and clinical signs will be assessed by in vivo analysis of gene knockout mice, and in horses by corticosteroid- blockade of Th1 immunity. Immune cell and cytokine profiles will be analyzed and correlated with pathology and clinical signs. The demonstration in animal models of the major pathways by which immune and inflammatory reactions induced by ehrlichiae cause pathologic lesions and clinical signs would be a major advance. Since in vitro growth modifies virulence, defining a linkage between bacterial modifications and immunopathologic reactions would provide a more complete understanding of the roles that antigenic and genetic variation play in the ehrlichioses or other infections. This information will provide the framework for rational design of new therapies aimed at modulating immune mechanisms and design of preventative measures aimed at immunization.